The role of lipid-modified proteins in cell wall synthesis and signaling

Abstract The plant cell wall is a complex and dynamic extracellular matrix. Plant primary cell walls are the first line of defense against pathogens and regulate cell expansion. Specialized cells deposit a secondary cell wall that provides support and permits water transport. The composition and organization of the cell wall varies between cell types and species, contributing to the extensibility, stiffness, and hydrophobicity required for its proper function. Recently, many of the proteins involved in the biosynthesis, maintenance, and remodeling of the cell wall have been identified as being post-translationally modified with lipids. These modifications exhibit diverse structures and attach to proteins at different sites, which defines the specific role played by each lipid modification. The introduction of relatively hydrophobic lipid moieties promotes the interaction of proteins with membranes and can act as sorting signals, allowing targeted delivery to the plasma membrane regions and secretion into the apoplast. Disruption of lipid modification results in aberrant deposition of cell wall components and defective cell wall remodeling in response to stresses, demonstrating the essential nature of these modifications. Although much is known about which proteins bear lipid modifications, many questions remain regarding the contribution of lipid-driven membrane domain localization and lipid heterogeneity to protein function in cell wall metabolism. In this update, we highlight the contribution of lipid modifications to proteins involved in the formation and maintenance of plant cell walls, with a focus on the addition of glycosylphosphatidylinositol anchors, N-myristoylation, prenylation, and S-acylation.


Introduction
The plant cell wall is a polysaccharide-rich extracellular matrix that surrounds cells.The composition and organization of the wall are highly dynamic, differing during development and between species and cell types (Gigli-Bisceglia et al. 2020).The primary cell wall is a relatively thin and flexible layer that supports cell expansion while resisting turgor pressure and protecting the cell against bacterial and fungal infection (Cosgrove 2005).In contrast, only specialized cells have a secondary cell wall, which provides the plant with structural support and allows water transport in xylem tissues (Bellincampi et al. 2014).Cell walls are also modified in response to biotic and abiotic stresses; these changes include the reinforcement of the wall to improve its performance as a barrier to microbes and changes that conserve energy and resources when light and nutrient conditions are limiting (Houston et al. 2016;Wang et al. 2016).Beyond being a protective layer, the cell wall is also the site of intracellular signaling, imposes a limitation on the diffusion of proteins at the plasma membrane, and contributes to the architecture of the cell (Zhong et al. 2019;Gigli-Bisceglia et al. 2020).
In angiosperms, primary cell walls are composed largely of cellulose, xyloglucan, and pectin, while the major components of secondary cell walls are cellulose, xylan, and lignin (Gigli-Bisceglia et al. 2020).Both types of cell wall also contain cell wall proteins.Despite making up less than 10% of the cell wall by mass, proteins are essential in cell wall structure and biogenesis.The roles of cell wall proteins include modifying polysaccharides, binding polysaccharides, and interacting with plasma membrane-localized receptors.These include proteins such as expansins (Cosgrove 2016;Narváez-Barragán et al. 2022), arabinogalactan proteins (AGPs) (He et al. 2019;Lin et al. 2022a, b, c) and pectin methylesterases (PMEs) (Shin et al. 2021).Many proteins localized within the cell wall and involved in the synthesis and delivery of cell wall components to the apoplastic space are modified with lipid groups.These lipid modifications include the addition of a glycosylphosphatidylinositol (GPI) anchor, N-myristoylation, prenylation, and S-acylation (Fig. 1).The function of each of these lipid modifications varies at least partly due to their targeting of proteins to different sides of the plasma membrane.GPI anchors tether proteins to the outer leaflet of the plasma membrane, whereas the other lipid modifications interact with the inner leaflet of the plasma membrane and membranous organelles (Giglione and Meinnel 2022;Xu et al. 2022aXu et al. , 2022b)).Lipid modification of many of these proteins is essential for their function in cell trafficking (Kumar et al. 2016;Rojek et al. 2021), membrane anchorage or interaction (Basu et al. 2016;Feng et al. 2018), protein-protein interaction (Mahajan et al. 2008), and membrane microdomain localization by promoting the clustering of similarly modified proteins to membrane regions with distinct lipid compositions (Xu et al. 2022a(Xu et al. , 2022b;;Stroppa et al. 2023).Evidence for the importance of lipid modification of proteins for plant cell wall synthesis and/or structure comes from studies that have disrupted the enzymes involved in lipid metabolism, which could be the underlying cause of resulting cell wall defects (Hemsley et al. 2005;Renna et al. 2013;Zhang et al. 2015;Bernat-Silvestre et al. 2021;Gutkowska et al. 2021).
Many proteins also bear multiple lipid modifications, being either dual lipid-modified or modified with the same modification at multiple sites.Dual lipid-modified proteins often rely on an irreversible modification such as prenylation or N-myristoylation to promote interactions with membranes, and a second modification such as S-acylation is required for localization and function (Li et al. 2022;Park et al. 2023).In this article, we summarize the roles of lipid modification of proteins in the regulation of cell wall deposition and its response to the environment.

GPI-anchored proteins
The attachment of proteins to the outer leaflet of the plasma membrane through C-terminal modification with a GPI anchor is conserved in eukaryotes.In plants, this results in protein localization to the apoplastic space.Recent genetic and proteomic studies predict that 248 Arabidopsis (Arabidopsis thaliana) proteins bear a GPI modification (Rui and Dinneny 2020).Following the attachment of the preassembled GPI anchor to a protein, the anchor undergoes further lipid and glycan remodeling, understood to be important for the processing of GPI-anchored proteins through the secretory system toward their final localization (Bernat-Silvestre et al. 2021).For some GPI-anchored proteins, the lipid modification can be cleaved, releasing the protein from the outer leaflet of the plasma membrane and allowing it to move into the apoplast (Sedbrook et al. 2002;Xue et al. 2017).Remodeling of the GPI anchor in the Golgi promotes clustering of similarly anchored proteins and aids their sorting during secretion to the apoplastic space (Xu et al. 2022a(Xu et al. , 2022b)).Currently, only 1 plant GPI anchor structure has been determined (Oxley and Bacic 1999).This structure, obtained from Pyrus communis suspension-cultured cells, confirmed that the core glycan is conserved between eukaryotes.Following the addition of a GPI anchor to a protein in the endoplasmic reticulum (ER), the GPI lipid moiety undergoes extensive remodeling; the steps of this are well established in yeast and mammals (Kinoshita et al. 2008;Kinoshita 2020).Many mammalian and yeast GPI lipid-modifying enzymes have orthologs in plants, and through studying these genes progress has been made in understanding GPI lipid remodeling in plants.In the Arabidopsis inositol deacylase mutant POST-GPI ATTACHMENT TO PROTEINS 1 (PGAP1), the exit of GPI-anchored proteins from the ER and delivery to the plasma membrane is delayed (Bernat-Silvestre et al. 2021).Interestingly, removal of the acyl group from the inositol ring by PGAP1 was shown to be required for the effective release of GFP-tagged SKU5 by cleavage of the GPI anchor of SKU5, suggesting that the lipid moiety of the GPI anchor is important for phospholipase recognition (Lin et al. 2022a(Lin et al. , 2022b(Lin et al. , 2022c)).Another GPI lipid remodeling enzyme, a membrane-bound O-acyltransferase, was identified as rice (Oryza sativa) Brittle Culm 16 (OsBC16) (Xu et al. 2022a,

ADVANCES BOX
• Genetic analysis has identified many diverse GPI-anchored proteins that play crucial roles in cell wall deposition during growth.These proteins include those that maintain a suitable environment in the apoplast for normal growth.
• The role of N-myristoylation and prenylation in cell wall biosynthesis is limited to the wellestablished functions of small GTPases that are important for polar secretion including that of cell wall components.
• Identification of an acylome facilitates the functional analysis of protein S-acylation by identifying which cysteines to mutate.
• Most if not all proteins involved in cellulose synthesis and the association of cellulose synthesis with microtubules are S-acylated, in contrast, only a small proportion of proteins involved in the synthesis of matrix polysaccharides are modified in this way.
• Several potential cell wall modifying enzymes are S-acylated, which likely regulates their localization.2022b).OsBC16 was found to be essential for targeting GPI-anchored OsBC1 to lipid nanodomains, where it functions in cellulose synthesis; this is likely due to the acyltransferase activity of OsBC16 during GPI lipid remodeling (Xu et al. 2022a(Xu et al. , 2022b)).
The essential role of GPI-anchored proteins in the regulation of cell wall structure and composition was demonstrated by peanut1 (pnt1) mutants caused by a defect in a homologue of PHOSPHATIDYLINOSITOL GLYCAN OF COMPLEMENTATION CLASS M (PIG-M).PIG-M is an endoplasmic reticulum-localized mannosyltransferase required for the synthesis of GPI anchors.pnt1 mutants exhibit reduced crystalline cellulose content, increased pectin deposition, and ectopic cell wall deposition and are seedling lethal (Gillmor et al. 2005).Understanding the role of GPI-anchored proteins in the cell wall is complicated by the large number of proteins modified in this way.While many GPI-anchored proteins appear essential for normal cell wall metabolism and signaling, many share only the anchor in common and represent a diverse set of protein structures (Fig. 2).Detailed reviews of GPI-anchored proteins are available (Desnoyer and Palanivelu 2020;Silva et al. 2020;Zhou 2022).Here we focus on selected examples that illustrate recurring themes regarding the role of GPI-anchored proteins in cell wall metabolism and signaling.

Arabinogalactan proteins (AGPs) and Fasciclin-like arabinogalactan proteins
AGPs comprise a large family of highly glycosylated, hydroxyproline-rich proteins that bear GPI anchors.They are localized in the apoplast, where they contribute to cell wall structure and function as signaling intermediates (Xue et al. 2017;Seifert 2018).One of the best-studied examples is the classical AGP, Arabinoxylan Pectin Arabinogalactan Protein 1 that integrates into the cell wall through covalent linkage of their arabinogalactan domains to cell wall hemicellulose and pectin (Tan et al. 2013).A subgroup of the AGPs also contains a Fasciclin-like (FAS) domain (Fig. 2).FAS domains are found in all kingdoms and act as cell adhesion domain in mammals, giving rise to the idea that Fasciclin-like AGPs (FLAs) function as putative sensors of cell wall integrity (MacMillan et al. 2010;He et al. 2019).Both expression and mutant data suggest FLAs are likely to possess additional roles in cell wall synthesis (see below).There are 21 FLAs in Arabidopsis and up to 50 in poplar, many of these arose due to gene duplication events and therefore redundancy likely masks FLA mutant phenotypes (Showalter et al. 2016); however, phenotypes associated with mutation in several FLA genes have been reported.
FLA4 was initially identified as Salt Overly Sensitive 5 during a screen for mutants with increased salt sensitivity and Structures and important features of lipid modifications.Glycosylphosphatidylinositol anchor modification is the addition of a glycolipid by amide linkage, at the "ω" site, following the cleavage of a C-terminal hydrophobic signal sequence.Various lipid and glycan structures exist; the glycosylphosphatidylinositol anchor shown is based on the structure determined in Oxley and Bacic (1999).Cleavage sites for phospholipase C (PLC) and D (PLD) are marked by arrows.N-myristoylation is the irreversible addition of a 14-carbon myristate acyl lipid group, by NMTs, through an amide linkage to a glycine residue at position 2 following the cleavage of the initiator methionine.Prenylation is the irreversible addition of 15carbon farnesyl (solid lines) or 20-carbon geranylgeranyl (dashed lines) isoprene lipid moieties through thioether linkage to C-terminal cysteines at various motifs, including CaaX, where "a" are aliphatic amino acids and "X" can be any amino acid.Alternatively, prenylation can occur in the absence of a CaaX motif by the interaction of the modified protein with REP.Multiple enzymes catalyze the addition of prenyl lipids; PFT, PGGT-I, and RabGGT.S-acylation is the addition of 16-carbon palmitate (solid lines) or 18-carbon stearate (dashed lines) acyl lipid to cysteine residues through a thioester linkage by PATs.S-acylation is not limited to protein N or C termini; there is little, or no, obvious consensus sequence around the modified cysteines.Unlike other lipid modifications, S-acylation is reversible through the action of APTs.
recognized as an AGP.FLA4 acts in a linear genetic pathway with a pair of functionally redundant leucine-rich repeats receptor-like kinases: FEI1 and FEI2 (Seifert et al. 2014;Basu et al. 2016).The salt-hypersensitive fla4 mutants have thinner cell walls, swollen root cells, and reduced root length following salt treatment (Shi et al. 2003).This phenotype is mirrored in the fei1/2 double mutant and is non-additive, with fla4, suggesting that they work in the same pathway.Growth of fla4 mutants is comparable to wild type under normal growth conditions, further suggesting that FLA4 acts during the response to salt stress; this is likely mediated by the predicted interaction of the C-terminal FAS domain of FLA4 with the leucine-rich receptor kinase FEI1 (Turupcu et al. 2018).FLA4 lacking the GPI anchor is largely retained in the ER.The fla4 mutant is complemented by FLA4 lacking the GPI anchor; this is likely because sufficient FLA4 is still able to reach the cell surface or is secreted.FLA4 likely acts predominantly within the cell wall, and its anchorage in the membrane probably promotes clustering with receptors or allows its controlled release into the wall by cleavage under specific circumstances, the details of which are not understood (Xue et al. 2017).Similarly, when the GPI modification is disrupted by a mutation in PGAP1, FLA11 is retained in the ER and Golgi (Bernat-Silvestre et al. 2021).The cell wall of the pgap1-3 mutant also has increased arabinan and reduced type II arabinogalactan and xyloglucan, emphasizing the potential role of GPI modification in both normal FLA11 targeting and cell wall construction (Bernat-Silvestre et al. 2021).
Overexpression of FLA11 resulted in premature secondary cell wall deposition, with the resultant cell wall having increased lignification and reduced crystalline cellulose content, similar to the typical changes in the cell wall following compression stress.This implicates FLA11 as a sensor of cell wall integrity, although it remains unclear how the absence of functional FLA11 results in reduced stem tensile strength (MacMillan et al. 2010;Ma et al. 2022).This large change in mechanical properties of fla mutants suggests a more fundamental role for FLA11/12 in determining cell wall structure (MacMillan et al. 2010).
Several other FLAs are GPI anchored and involved in the regulation of the cell wall during development (Johnson et al. 2003;Liu et al. 2020).FLA3 disruption by RNA interference results in aberrant cellulose deposition in the pollen intine cell wall as well as shrunken and wrinkled pollen grains (Li et al. 2010).Similar pollen tube growth phenotypes are seen in mutants of 2 subunits of the N-acetylglucosaminyltransferase required for the synthesis of the GPI anchor, suggesting that the GPI anchor of FLA3 is important for its function, potentially by promoting the clustering of GPI-anchored receptors at the plasma membrane (Lalanne et al. 2004).

Pectin modification by GPI-anchored enzymes
Pectins are a diverse group of polysaccharides with a common core of 1,4-linked α-D-galacturonic acid found predominantly in the primary cell wall, where they contribute to the formation of a gel-like matrix surrounding the cellulose fibrils (Voragen et al. 2009;Shin et al. 2021).Synthesized in the  (Adebali et al. 2015).CBM2, carbohydrate-binding type-2; GDPD, glycerophosphodiester phosphodiesterase; Glyco hydro 17, Glycoside hydrolase family 17; The ω sites predicted using PredGPI are marked in red (Pierleoni et al. 2008).
Golgi, pectins are secreted into the apoplastic space, where they are further modified, altering the porosity and surface charge of the wall (Shin et al. 2021).Newly synthesized homogalacturonan domains of pectin can be demethylated by pectin methylesterases (PMEs), resulting in increased cell wall flexibility that has been shown at the shoot apical meristem and is likely to be a feature of many cell types (Braybrook and Peaucelle 2013).Numerous PMEs and PME inhibitors (PMEIs) are predicted to bear GPI anchors.Currently, only PMEI1 has been experimentally confirmed to be GPI anchored, with GPI modification potentially facilitating its Golgi exit and secretion to the apoplast.The requirement for the GPI anchor of PME1 has not yet been experimentally validated (De Caroli et al. 2011).To date, the most likely potential function of GPI anchoring of PMEs and PMEIs is for their sorting and secretion to the outer leaflet of the plasma membrane; however, it is possible that GPI anchors could promote clustering of PME and PMEI pairs to fine-tune pectin structure in response to developmental or stress cues (Braybrook and Peaucelle 2013;Wu et al. 2018).
Other pectin-modifying enzymes are also predicted to be GPI anchored.Pectin lyases cleave the homogalacturonan backbone of pectin, preferentially acting on regions that have been demethylesterified by the action of PMEs (Wang et al. 2018).Pectin Lyase-Like 13 was initially identified as Powdery Mildew Resistance 6 (PMR6), with the pmr6-1 phenotype displaying smaller rosette leaves, increased pectin content, and increased pectin demethylesterification (Vogel et al. 2002;Sun and van Nocker 2010).As for PMEI1, the requirement for GPI anchorage of PMR6 has not been demonstrated.Potential roles for the GPI anchor of PMR6 would include contributing to the localized release of pectin hydrolysis products that act as signaling intermediates (Hernandez-Blanco et al. 2007).The small size of pmr6-1 plants implicates PMR6 in normal growth and development, although the function and requirement of the putative GPI anchor warrants further investigation.

GPI-anchored cell wall sensors
An important function of plant GPI-anchored proteins is to support the transduction of signals regarding the status of the cell wall into the cell.This is typified by the interaction of 2 plant GPI-anchored proteins, LORELEI (LRE) and LRE-like GPI-Anchored Protein 1 (LLG1), with the extensively studied Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase FERONIA (FER) (Liu et al. 2016a, b;Feng et al. 2018).LRE and LLG1 interact directly with FER in the apoplast, activating distinct signaling cascades at different developmental stages and in response to changes in environmental conditions.The attachment of a GPI anchor to LRE is essential for its localization to the outer leaflet of the plasma membrane in the filiform apparatus but not for its function during pollen tube reception (Liu et al. 2016a(Liu et al. , 2016b)).FER is involved in sensing the softening of the cell wall under high salt conditions, triggering multiple calcium signaling pathways, including the release of Ca 2+ into the wall to reinforce the cell wall matrix via pectin-Ca 2+ crosslinking (Feng et al. 2018).Intriguingly, FER was recently revealed to directly bind to demethylesterified pectin in the cell wall and regulate signaling via Rho-like GTPases, Rho of plants (ROP) 2, 6, and 11.These ROPs are also S-acylated and prenylated (see below), implying that multiple lipid modifications are important for different steps in the plant cell wall integrity and stress signaling (Chai et al. 2016;Sugiyama et al. 2019;Lin et al. 2022aLin et al. , 2022bLin et al. , 2022c)).

COBRA and COBRA-like families
The plant-specific COBRA (COB) and COBRA-like (COBL) genes encode GPI-anchored proteins that are essential for cell wall biosynthesis, regulating cellulose crystallinity and contributing to cell wall expansion and anisotropic growth (Roudier et al. 2002;Brady et al. 2007).The founding member of the COB family was identified due to its mutant displaying abnormal root expansion and reduced crystalline cellulose with increased amounts of disorganized cellulose (Benfey et al. 1993;Schindelman et al. 2001).Many COB and COBL genes are coexpressed with cellulose biosynthesis genes, owing to their function in binding to cellulose during its deposition in the cell wall (Brown et al. 2005;Brady et al. 2007;Ben-Tov et al. 2018).Investigation of COB and COBL function has been carried out extensively in crop species.Rice (Oryza sativa) BRITTLE CULM 1 (OsBC1), a homologue of COBL4, exhibits abnormal secondary cell wall deposition with reduced cellulose and increased lignin content, resulting in their characteristic brittle culms (Sato-Izawa et al. 2020).OsBC1 interacts with crystalline cellulose through its carbohydrate-binding domain and is anchored in the plasma membrane by its GPI anchor (Liu et al. 2013).The GPI anchor can be cleaved to release OsBC1 into the cell wall; however, the significance of this cleavage is still unclear (Fig. 3) (Liu et al. 2013).Mutation of OsBC16, involved in GPI lipid anchor synthesis, results in the same reduced cell wall elasticity, fragile internodes, and reduced cellulose content as OsBC1 and is essential for GPI modification of OsBC1.Loss of OsBC16 also disrupts OsBC1 targeting to the plasma membrane and membrane nanodomains, which is likely important for its interaction with cellulose in the cell wall and implicates GPI anchoring in OsBC1 function (Xu et al. 2022a(Xu et al. , 2022b)).Further evidence of the importance of the GPI anchor in targeting proteins to particular regions of the plasma membrane comes from COBL10.GPI anchorage of COBL10 is essential for its localization to the apical tip of pollen tubes, where it functions in cell wall organization, likely by regulating the pectin matrix and cellulose orientation required for female-signal guided expansion of the pollen tube (Li et al. 2013).Genetic analysis has identified an essential role for other COBL proteins in diverse cell wall types.Mutation in roothairless 3 (rth3), a maize COBL gene family member, disrupts cell wall deposition during root hair development (Hochholdinger et al. 2008;Li et al. 2022), and cobl2 has defective seed coat mucilage polysaccharide organization (Ben-Tov et al. 2018).

GPI-anchored proteins as coordinators of multicellular growth
The embryo-lethal pnt1 mutant (see above) results from a defect in the synthesis of the GPI anchor (Gillmor et al. 2005).While embryos exhibit clear cell wall defects, callus derived from the mutant exhibits none of these defects.This observation led the authors to suggest that proteins with GPI anchors may be particularly important in coordinating multicellular growth.Some support for this suggestion comes from studies on mutants in several GPI-anchored proteins.Mutants in both ZERZAUST, a protein with homology to β-1,3 endoglucanases, and SKU5 cause the twisting of organs, consistent with defective coordination between cell layers (Fulton et al. 2009;Vaddepalli et al. 2017).The triple mutant between SKU5 and two related GPI-anchored proteins SKU5-SIMILAR1 (SKS1) and SKS3 exhibit huge variation in cell size even between adjacent cells (Sedbrook et al. 2002;Zhou 2019).It is unclear how these GPI-anchored proteins coordinate cell growth, but they do illustrate very clearly the link between proper cell wall synthesis and cell expansion.It is possible that GPI-anchored proteins, such as SKU5, generate signals that are perceived by adjacent cells.SKU5 is characterized by 3 domains exhibiting homology to blue copper oxidases (Fig. 2); however, it lacks several conserved amino acids likely to be essential for activity and consequently.Any enzymatic function and any signal this might generate remain unknown.More recently SKU5 and SKS1 have been shown to modulate ROS-based signals generated by NADPH oxidase, and this modulation is essential for cell wall structure and root growth.The GPI anchor was absolutely essential for the function and targeting of SKU5 and SKS1 to the plasma membrane (Chen et al. 2023).
Unfortunately, due to space limitations, we could not include the details of many other genes encoding GPI-anchored proteins with novel functions in the cell wall.Other examples of GPI-anchored proteins with known or putative roles in cell wall biosynthesis and regulation of which have also recently been reviewed (Zhou 2022).

N-myristoylation and the cell wall
Protein N-myristoylation is the addition of a saturated 14-carbon fatty acid moiety to the alpha-amino group of the N-terminal glycine residue of a protein after the removal of the initiator methionine by a methionine aminopeptidase (Giglione et al. 2000).It is also possible that myristoylation occurs at internal glycine residues following protein cleavage that reveals a newly exposed terminal glycine, as has been shown for human proteins (Thinon et al. 2014;Majeran et al. 2018).The myristate group is added by the formation of an amide linkage to the glycine (Fig. 1), and currently, there is no evidence that the myristate group is removed in plants.However, the myristoyl group can be masked in various ways to regulate its membrane association (Matheson et al. 2008;Giglione and Meinnel 2022).N-myristoylation supports membrane interaction but is not sufficient for stable membrane association and therefore acts as a substrate for further lipid modifications such as S-acylation by membrane-bound DHHC-type protein acyl transferases (PATs) (Batistic 2012;Castrec et al. 2018).In Arabidopsis, there are 2 N-myristoyltransferases (NMTs), and knockout of the more active, NMT1, is lethal, whereas nmt2 mutants display defects in the shoot apical meristem during embryonic development, aberrant cell polarity, and growth arrest, implicating N-myristoylation in multiple developmental processes that require appropriate cell wall maintenance and deposition (Pierre et al. 2007;Renna et al. 2013).

Myristoylation of SOS3 for cell wall integrity maintenance during salt stress
Perception of salt stress is important for plants to mount appropriate responses to maintain their growth and development.Sensing salt concentrations occurs at the cell wall, and signals generated in the wall are transduced into the cell via plasma membrane-localized receptors, such as described for GPI-anchored proteins FLA4 and LRE/LLG1.Salt overly sensitive 3/calcineurin B-like protein (SOS3/CBL4) is an N-myristoylated Ca 2+ sensor that activates SOS2, a protein kinase required for salt tolerance by maintaining normal Na + / K + concentration within the cell (Liu et al. 2000).Together SOS2/3 activate the Na + /H + antiporter, SOS1, at the plasma membrane, resulting in the transport of Na + out of the cell.N-myristoylation of SOS3 is required for the calciumdependent interaction of SOS3 with SOS2 and is also required for salt tolerance (Fig. 3) (Mahajan et al. 2008).Here, the coupling of Na + export from the cytosol and H + import could contribute to the maintenance of cell wall pH to prevent acidification-related loosening of the cell wall by expansins, thus maintaining cell wall integrity (Cosgrove 2016;Colin et al. 2023).Similarly, extracellular pH is also coordinated with cellulose synthesis during cell expansion by the GPI-anchored protein SHAVEN3 and its homolog, SHAVEN3-like 1 (Hayashi et al. 2008;Yeats et al. 2016).

Maintenance of polarized cell wall deposition at root hair tip by CBLs
Root hairs are long and thin extensions of root epidermal cells, and their formation requires the polarized extension of the cell by deposition of new cell wall material at the apex of the elongating root hair.This extension requires apoplastic accumulation of reactive oxygen species (ROS) and Ca 2+ gradients for the appropriate cell wall loosening for expansion and for the localized secretion of cell wall components (Zhang et al. 2022).Like SOS3/CBL4, other CBLs, CBL1, 5, and 9, are dual-lipid modified with N-myristoylation and S-acylation.Dual lipid modification is required for CBL1 localization to the plasma membrane (Batistic et al. 2008).CBL1 contributes to the membrane lipid environment, Ca 2+ gradients, and ROS production, all of which are required for the polar delivery of cell wall components at the tip of root hairs (Fig. 3) (Preuss et al. 2006;Zhang et al. 2022).

Prenylated proteins at the plant cell wall
Prenylation is the irreversible addition of polyisoprene lipids to cysteine residues at the C terminus of proteins; typically 15-carbon farnesyl or 20-carbon geranylgeranyl units are added (Fig. 1).Like other lipid modifications, prenylation introduces a hydrophobic group that contributes to the interaction of the modified protein with specific cellular membranes.Farnesyl and geranylgeranyl moieties are added through thioether linkages by protein farnesyl transferase (PFT) and protein geranylgeranyl transferase (PGGT-I).These prenyltransferases are heterodimeric, with a common α subunit and β subunits that confer protein specificity and determine modification by farnesyl or geranylgeranyl lipid (Hemsley 2015).Prenylation by PFT and PGGT-I occurs at the cysteine residue of the CaaX box consensus at the protein C terminus, where "a" represents aliphatic amino acids and "X" represents any amino acid that depends on the substrate.Following prenylation, the aaX peptide is removed, and the newly freed carboxyl group of the new terminal cysteine is methylated (Fig. 1) (Chang et al. 2021).

Dual geranylgeranyl modification of Rab GTPases
In addition to PFT and PGGT-I, there exists a Rab GTPase-specific geranylgeranyl transferase, RabGGT, that appears to modify only Rab family members, acting only on Rab GTPases in complex with Rab Escort Protein (REP).Twin geranylgeranyl modification of Rab GTPases is less reliant on the CaaX motif, and instead, RabGGT specificity is imposed by interaction with REP, prenylating both cysteines at XCC, XCXC, XCCX, CCXX, and CCXXX motifs (Shi et al. 2016).Rabs are small GTPases involved in vesicular transport and function in many plant-specific signaling pathways involving cell wall regulation and pathogen defense (Gutkowska et al. 2021).Many of the components of the cell wall, such as hemicelluloses and pectins, are synthesized by enzymes in the endomembrane system before their eventual targeted release into the cell wall (Kim and Brandizzi 2014;Meents et al. 2019).Membrane cycling of Rab GTPases is critical to their interaction with their respective effectors and the resulting appropriate vesicular transport implicating them in multiple cell wall-related processes (MartiniÈre and Moreau 2020).In Arabidopsis, global disruption of Rab geranylgeranyl modification by mutation of the RGTB1 gene, encoding the RGTβ subunit, results in pollen tube and root hair defects, reminiscent of those seen for mutants of N-myristoylated and S-acylated CBL1 (Batistic et al. 2008;Gutkowska et al. 2015).This is likely a result of defects in the endocytic and exocytic vesicular trafficking needed to maintain polarized cell expansion at tipgrowing cells (van de Meene et al. 2017).
Cellulose synthesis is partially regulated by trafficking cellulose synthase complexes (CSCs) to and from the plasma membrane, where they are active in cellulose biosynthesis (Zhu and McFarlane 2022).RabH1b has recently been implicated in this cycling whereby disruption of the RabH1b gene results in reduced motility of the primary cell wall CELLULOSE SYNTHASE A6 at the plasma membrane (Fig. 3) (He et al. 2018).Although RabH1b is not currently confirmed to be prenylated, it does have appropriately situated cysteines for recognition by REP.Similarly, RabA4b is putatively prenylated and is identified as an interactor of phosphatidylinositol 4-OH kinase, PI4Kβ1, potentially implicating it in the regulation of membrane lipid at the trans-Golgi network (Fig. 3) (Preuss et al. 2006;Jia et al. 2018;Zhou et al. 2020).The role of the RabA4 subfamily in tip-growing cells is also illustrated by the requirement for RabA4d for pollen tube growth (Szumlanski and Nielsen 2009).Overall, Rab GTPases require prenylation for their membrane interaction and function and even have their own prenyltransferases.However, it is still unclear whether the phenotypes associated with Rab mutants are due to global defects in vesicular transport or can be attributed directly to the transport of cell wall related enzymes and cargoes.

Prenylated ROPs
In the moss Physcomitrella patens, knockout of the prenyltransferases PpGGTβ subunit has been implicated in cell-cell adhesion through the maintenance of cell wall integrity that is required to maintain multicellularity (Bao et al. 2022).The PpGGTβ loss of multicellularity and polar cell elongation phenotype was rescued by the introduction of the Arabidopsis PGGT-I, indicating that the geranylgeranyl transferase function is conserved (Thole et al. 2014).The phenotype of PpGGTβ has since been linked to the geranylgeranyl modification of ROP GTPases (Bao et al. 2022).Indeed, ROP involvement in cell wall biosynthesis is well documented in root hair growth, cell wall status monitoring, and secondary cell wall patterning through the regulation of microtubule dynamics (Chai et al. 2016;Feiguelman et al. 2022;Lin et al. 2022aLin et al. , 2022bLin et al. , 2022c;;Xu et al. 2022aXu et al. , 2022b)).Type II ROPs (ROP9, 10, and 11) are S-acylated, and Type I ROPs (ROP2, 4, and 6) are prenylated, although some type I ROPs are dual modified, bearing both prenylation and S-acylation (Li et al. 2022).

S-acylation of cell wall-related proteins
S-acylation, or palmitoylation, as it is commonly known, is the post-translational addition of a fatty acid moiety, usually stearate or palmitate in plants, to a cysteine residue (reviewed in Chamberlain and Shipston 2015;Hurst and Hemsley 2015;Hemsley 2017;Li and Qi 2017;Zheng et al. 2019;Hemsley 2020;Wang and Yang 2021;Li et al. 2022).The hydrophobic nature of the fatty acid molecules means that S-acylation, like other lipid modifications of proteins, increases the membrane affinity of the modified proteins.For cytosolic proteins, S-acylation results in membrane association; however, S-acylation is also known to occur in proteins that possess transmembrane helices.For these transmembrane proteins, the roles of S-acylation include protein targeting to specific membrane microdomains, protein trafficking, protein-protein interactions, tilting of transmembrane helices, and protein stability (Charollais and Van Der Goot 2009;Chamberlain and Shipston 2015).Unlike other lipid modifications of proteins, S-acylation is freely reversible.
Several hundred plant proteins were identified to be S-acylated using global proteomic approaches (Hemsley et al. 2013;Srivastava et al. 2016).However, further progress was hampered due to a lack of information on the exact sites of S-acylation.This knowledge gap was addressed in a recent study that identified 1849 S-acylated cysteines located within 1094 proteins and estimated that around 6% of all proteins are modified by S-acylation (Kumar et al. 2022).Although S-acylated proteins are involved in many aspects of plant metabolism and signaling, modification by S-acylation features prominently among proteins involved in cell wall biosynthesis and cellulose synthesis in particular.

Cellulose synthesis
CELLULOSE SYNTHASE A (CESA) proteins, the catalytic subunits of the CSC, are among the most heavily modified proteins by S-acylation, with up to 8 cysteines modified in different CESA proteins.In Arabidopsis CESA7, 4 cysteines in a region known as variable region 2 (VR2) and 2 cysteines in the C-terminus (CT) are modified by S-acylation, and these are important for plasma membrane localization of the CSC (Kumar et al. 2016).A subsequent global proteomic study confirmed that VR2 and/or CT cysteines are S-acylated in both primary and secondary cell wall CESA proteins (Kumar et al. 2022).However, sites outside of these regions were also S-acylated in some CESA proteins, pointing to functional diversification of some protein domains between different CESA proteins.In particular, the cysteines located toward the amino terminus, within a putative RING finger domain and assumed to chelate zinc, were identified to be S-acylated for CESA8.Modification of these cysteines by S-acylation is not compatible with a role in zinc binding.These data suggest that, although the RING domain of all CESA proteins contain 8 conserved cysteines, there is structural, and probably functional, divergence between the domains from CESA7/CESA4/CESA8.Many other proteins involved in cellulose synthesis were also identified as being S-acylated (Fig. 4) (Kumar et al. 2022).These include proteins that colocalize with the CSC, such as KORRIGAN (Vain et al. 2014), COMPANION OF CELLULOSE SYNTHASE 1 (Endler et al. 2015), and CELLULOSE SYNTHASE INTERACTING (CSI) (Gu et al. 2010); proteins that mediate CSC trafficking, such as SHOU4, SHOU4L (Polko et al. 2018), TRANVIA (Vellosillo et al. 2021), and7TM5 (McFarlane et al. 2021); in addition to proteins that are involved in exocytosis or endocytosis of the CSC, such as AP2M (Bashline et al. 2013) and EXO84B (Gu and Rasmussen 2022).The function of S-acylation of these proteins is not entirely clear; however, it is known that S-acylation can promote the clustering of membrane proteins.The highly modified nature of the CSC and the very hydrophobic environment this creates may mean that only proteins that are S-acylated and similarly hydrophobic are able to associate with the CSC.
Microtubules play a crucial role during cellulose synthesis, where they are involved in both the delivery of CSCs to the plasma membrane and guiding CSC movement through the plasma membrane.Tubulin proteins, the structural components of microtubules; chaperonin containing T-complex polypeptide-1 complex, a multi-subunit chaperonin complex that is involved in tubulin folding (Ahn et al. 2019); and CELLULOSE MICROTUBULE UNCOUPLING (CMU) proteins that anchor the cortical microtubules to the plasma membrane (PM) (Liu et al. 2016a(Liu et al. , 2016b) ) are all modified by S-acylation (Hemsley et al. 2008;Kumar et al. 2022).Although none of these proteins contain transmembrane helices, some, like CMU, are known to localize to membranes.It is likely that S-acylation will contribute to their membrane localization and probably their association with sites of cellulose synthesis.

Matrix polysaccharides
Proteins involved in the biosynthesis of other major cell wall polysaccharides, hemicelluloses and pectins are also modified by S-acylation (Kumar et al. 2022).Compared with cellulose biosynthesis, far fewer biosynthetic proteins for noncellulosic polysaccharides are modified by S-acylation.For example, among the xylan biosynthesis proteins, only GlucUronic acid substitution of Xylan 1, which is involved in the addition of glucuronic acid and 4-O-methyl glucuronic acid sidechains to xylan (Mortimer et al. 2010), is S-acylated (Kumar et al. 2022).Similarly, for xyloglucan biosynthesis, only MUR3, a galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan (Madson et al. 2003), is S-acylated.Arabidopsis MUCILAGE-RELATED10 (MUCI10) and closely related paralog GLYCOSYLTRANSFERASE 6, are both S-acylated.MUCI10 has been proposed to be involved in the decoration of glucomannan chains (Voiniciuc et al. 2015), which are synthesized by the action of CELLULOSE SYNTHASE-LIKE A2.COTTON GOLGI-RELATED 2 (CGR2) and CGR3 play an important role in the methylesterification of pectins (Kim et al. 2015), and both proteins are S-acylated.It is interesting to note that all hemicellulose and pectin biosynthetic proteins that are S-acylated act on the side chains of the polysaccharides rather than the backbone.It is possible that this may reflect the organization of hemicellulose and pectin biosynthesis pathways within the Golgi with S-acylation targeting proteins to where they are best localized to affect later steps in the biosynthetic pathway.
Additionally, several hemicellulose and pectin modification and degradation enzymes are also S-acylated.This includes XYLANASE1, which is involved in xylan degradation; BETA-GALACTOSIDASE 10, xyloglucan endotransglucosylase/hydrolases XTH31 and XTH32 involved in xyloglucan modification and degradation; and a number of proteins that are involved in pectin modification and degradation like PECTIN METHYLESTERASE 16 (PME16), PME inhibitor PMEI-PME51, beta galactosidases BGAL1, BGAL2, BGAL6, BGAL8, BGAL9, beta-D-xylosidases BXL4, BXL6, and pectin acetylesterases PAE5, PAE7, PAE8 and PAE12 (Kumar et al. 2022).All of these enzymes are localized to the apoplast, so the presumed role of S-acylation for these enzymes would be in their trafficking to the extracellular space via exocytosis.

Lignin biosynthesis
Biosynthesis of monolignols, the building blocks of lignin, takes place at the interface of the cytosol and ER membrane.Several enzymes catalyzing the formation of monolignol subunits are modified by S-acylation, including PHENYLALANINE AMMONIA-LYASE, 4-COUMARATE: COA LIGASE, CAFFEOYL COENZYME A ESTER O-METHYLTRANSFERASE, CINNAMOYL COA REDUCTASE, CAFFEATE O-METHYLTRANSFERASE, and CINNAMYL ALCOHOL DEHYDROGENASE (Kumar et al. 2022).None of these monolignol biosynthesis enzymes possess a transmembrane helix, and therefore S-acylation could provide a mechanism for the transient anchoring of these enzymes to ER membranes.

Cell wall integrity sensing
Cell wall integrity-sensing mechanisms involve protein sensors that detect cell wall damage or other mechanical cues and activate the downstream signaling events.FEI1 is one such sensor that belongs to the Leucine-rich repeat receptorlike kinase family and is plasma membrane localized.FEI1 interacts with the GPI-anchored protein FLA4 but is also S-acylated (Kumar et al. 2022).S-acylation of a receptor kinase, FLS2, has recently been shown to be important for receptor complex activation in response to the binding of its ligand (Hurst et al. 2023), and S-acylation may serve a similar role with FEI1.Whether S-acylation may target FEI1 to membrane domains where it is able to colocalize with GPI-anchored proteins in the same pathway is unclear.MID1-COMPLEMENTING ACTIVITY1 (MCA1), a mechanosensitive calcium channel (Nakagawa et al. 2007), is also S-acylated.MCA1 functions downstream of Theseus1, another integrity sensor that belongs to the CrRLK1L subfamily of protein kinases.MCA1 has been suggested to be involved in the production of jasmonic acid and salicylic acid in seedlings treated with the cellulose synthase inhibitor isoxaben (Denness et al. 2011;Engelsdorf et al. 2018).

(De-)S-acylation enzymes and dynamics
S-acylation is a reversible modification with the addition of the fatty acid catalyzed by PATs and its removal by APTs.In Arabidopsis, there are 24 PAT enzymes that are localized to various subcellular spaces (Batistic 2012).In animal species, de-S-acylation is catalyzed by 1 to 2 serine hydrolases (APT1/2) (Dekker et al. 2010) and Alpha/Beta Hydrolase Domain-containing 17A/B/C (ABHD17) proteins (Lin and Conibear 2015).Although the homologs of APT1/2 have not been identified in plants, 11 members of the Arabidopsis ABHD17-like acyl protein thioesterases family were reported to exhibit thioesterase activity (Liu et al. 2021).
Cycling of a modified protein between S-acylated and de-S-acylated states, by the action of PATs and APTs, offers a powerful and dynamic regulatory mechanism for protein function.Although it is possible to see the effects of myristoylation by altering the protein structure to mask the modification, S-acylation is reversible in a manor analogous to that of other common protein modifications, such as phosphorylation (Wang et al. 2017).
Although future studies are likely to reveal more information on how the reversibility of lipid modification is important for proteins cycling between the plasma membrane and the cytosol, current examples of (de-)S-acylation enzyme/ substrate pairs or dynamic regulation of S-acylation are rare in plants.One notable exception is SGN1, a receptor-like cytoplasmic kinase (RLCK) that belongs to the RLCK VII family.It is required for proper positioning and formation of Casparian strip (Alassimone et al. 2016).Although SGN1 is localized to the plasma membrane, it does not contain any TM helices with S-acylation being responsible for its plasma membrane attachment.In the presence of the S-acylation inhibitor, 2-bromo-palmitate, SGN1 accumulates in the cytosol, even if the samples have been pre-treated with protein synthesis inhibitor, cycloheximide.These observations suggested that SGN1 undergoes cycles of S-acylation and de-S-acylation resulting in repeated attachment and de-attachment to the plasma membrane, helping establish polarity of SGN1 and consequent proper localization of the Casparian strip within the cell wall (Alassimone et al. 2016).

S-acylation perspectives
Identification of an S-acylated protein and the potential cysteines at which it might be modified is only the first step in the investigation of the role of S-acylation in the function of a particular protein and the biological process it is involved in.This is followed by mutation of target cysteines and studying the effect of loss of S-acylation on the cell biology and/or biochemistry of the target protein, thereby establishing a more precise role of S-acylation in a given process.Among the cell wall-related proteins, so far, this kind of analysis has only been performed for a small number of proteins like CESA proteins (Kumar et al. 2016) and KORRIGAN (Kumar et al. 2022).The availability of the comprehensive acylome dataset with the acyl-site information has the potential to greatly facilitate such studies.
It is important to establish a full complement of substrates for the PATs and the APTs.Given that there are more than 1000 S-acylated proteins and only 24 PATs and 11 APTs, each of the PAT and APT is likely to act on dozens, if not hundreds, of substrate proteins.Although PAT/APT substrates have been identified (Liu et al. 2021), further studies involving novel technologies, such as proximity labelling and high throughput proteomics, will be required to establish the identity of those substrates and untangle the function of individual PAT and APT enzymes.The ultimate goal would be to establish the regulatory network where all components involved in S-acylation and de-S-acylation of a target protein are known.Only then will we be able to establish how S-acylation modifies protein function in response to endogenous and exogenous cues.

Concluding remarks
The post-translational modification of proteins with a lipid moiety has functions in protein-protein interaction, targeting proteins to specific membranes and regions within membranes.Through these functions, lipid-modified proteins have roles in communicating the cell wall status into the cell as well as controlling normal cell wall deposition during development and in response to biotic and abiotic stress.N-myristoylation, prenylation, and S-acylation are involved in many similar functions.In some instances, this is not surprising because both prenylation and N-myristoylation can precede modification by S-acylation (see above), although these modifications involve proteins localizing to the endomembrane and the inner leaflet of the plasma membrane.In contrast, GPI-anchors tether proteins to the plasma membrane but outside of the cell.Both S-acylation and GPI-anchors have the potential to support the clustering of cell wall synthesis enzymes and integrity receptors.However, they operate on opposite sides of the plasma membrane.Whether these modifications function entirely independently is unclear because there is no information regarding any similarities between protein clustering driven by S-acylation or the addition of a GPI-anchor.The potential for inter-leaflet communication and organization of proteins within both leaflets of the plasma membrane would be an interesting avenue for future research.
An increasingly large number of GPI-anchored proteins have been identified as being essential for normal cell wall deposition.Many of these proteins share little in common other than the GPI anchor.In many cases, it remains unknown how they function to alter the cell wall (see "Outstanding Questions").The fact that these GPI-anchored proteins are likely to localize on the outer leaflet of the plasma membrane highlights the importance of the environment in the region immediately adjacent to the plasma membrane as being of central importance to normal cell wall deposition, but one that is very sensitive to external signals and perturbations.GPI-anchored proteins that apparently regulate cell wall deposition and integrity sensing, and coordinate multicellular growth, reflect the fact that cell wall deposition must be sensitive to feedback from inside the cell, the cell wall, adjacent cells, and potential biotic interactions.A diverse set of GPI-anchored proteins are important for all these processes (Fig. 2), and the GPI anchor is responsible for their targetting to the outer leaflet of the plasma membrane, where they are best positioned to perceive these signals and interact with other cell surface receptors as well as components of the cell wall biosynthesis machinery such as the CSC.
Although N-myristoylated, prenylated, and S-acylated proteins also exhibit lipid/fatty acid modification, they share very little in common with GPI-anchored proteins with the former having the attached lipid group and at least part of the protein remaining within the cell.The role of prenylated and N-myristoylated proteins in cell wall deposition is limited.However, these modifications have a well-characterized role in modifying small GTPases.These small GTPases are

OUTSTANDING QUESTIONS BOX
• To what extent does the structure of GPI anchors vary, and is variation in the structure of the GPI anchor important in the localization of GPI-anchored proteins within the plasma membrane and therefore cell wall?
• How do GPI-anchored proteins function to coordinate multicellular growth?
• Why are so many structurally diverse GPI-anchored proteins important for normal cell wall synthesis during growth?
• Why is the targeting of GPI-anchored proteins to membrane domains important for their function?
• Does the S-acylation of cell wall biosynthesis proteins promote their interaction during cellulose synthesis?
• Does S-acylation promote enzyme association in the ER and substrate channeling during monolignol biosynthesis?
• Is the reversible nature of S-acylation important for cell wall biosynthesis and could this regulate proteins that control the secretion of cell wall modifying enzymes?important for polarized vesicle targeting and are consequently particularly important in processes such as tip growth.Given that around 6% of proteins are modified by S-acylation, it is not surprising that some proteins related to cell wall biosynthesis are modified in this way, however, the fact that most if not all proteins related to cellulose synthesis are modified in this way and the particularly large number of acyl groups on the CSC suggests that it plays a particularly important role in cellulose synthesis.S-acylation is known to promote protein-protein interactions, and potentially this may be its role in the cell.Whether this is the result of the CSC localizing in specialized membrane domains is unknown.S-acylation of other proteins such as those involved in matrix polysaccharide or lignin biosynthesis may also promote the formation of enzyme complexes involved in substrate channeling (see "Outstanding Questions").

Figure 1 .
Figure 1.Structures and important features of lipid modifications.Glycosylphosphatidylinositol anchor modification is the addition of a glycolipid by amide linkage, at the "ω" site, following the cleavage of a C-terminal hydrophobic signal sequence.Various lipid and glycan structures exist; the glycosylphosphatidylinositol anchor shown is based on the structure determined inOxley and Bacic (1999).Cleavage sites for phospholipase C (PLC) and D (PLD) are marked by arrows.N-myristoylation is the irreversible addition of a 14-carbon myristate acyl lipid group, by NMTs, through an amide linkage to a glycine residue at position 2 following the cleavage of the initiator methionine.Prenylation is the irreversible addition of 15carbon farnesyl (solid lines) or 20-carbon geranylgeranyl (dashed lines) isoprene lipid moieties through thioether linkage to C-terminal cysteines at various motifs, including CaaX, where "a" are aliphatic amino acids and "X" can be any amino acid.Alternatively, prenylation can occur in the absence of a CaaX motif by the interaction of the modified protein with REP.Multiple enzymes catalyze the addition of prenyl lipids; PFT, PGGT-I, and RabGGT.S-acylation is the addition of 16-carbon palmitate (solid lines) or 18-carbon stearate (dashed lines) acyl lipid to cysteine residues through a thioester linkage by PATs.S-acylation is not limited to protein N or C termini; there is little, or no, obvious consensus sequence around the modified cysteines.Unlike other lipid modifications, S-acylation is reversible through the action of APTs.

Figure 2 .
Figure2.Domain organization of a selection of GPI-anchored proteins involved in cell wall regulation.A diagrammatic representation of the domain architecture of several GPI-anchored proteins involved in cell wall-related processes.Domain organization is based on protein sequences searched using CDvist(Adebali et al. 2015).CBM2, carbohydrate-binding type-2; GDPD, glycerophosphodiester phosphodiesterase; Glyco hydro 17, Glycoside hydrolase family 17; The ω sites predicted using PredGPI are marked in red(Pierleoni et al. 2008).

Figure 3 .
Figure 3. Lipid-modified proteins in cell wall integrity signaling, biosynthesis, homeostasis, and salt stress perception.Representative examples to illustrate the diverse roles of lipid-modified proteins in regulating cell wall properties and signaling.The prenylated Rab GTPases, RabA4b and RabH1b, coordinate the trafficking of PI4Kβ1 and the CSC, respectively.A rice COBRA-like protein, OsBC1 has a cleavable GPI anchor, allowing its release into the cell wall.Increased cytosolic Ca 2+ acts as a second messenger during salt stress and with N-myristoylation promotes the plasma membrane localization of SALT OVERLY SENSITIVE 3/CALCINEURIN B-LIKE PROTEIN 4 (SOS3/CBL4) and for interaction and activation of Na+/H+ antiporter SOS1 by SOS2/CBL-INTERACTING PROTEIN KINASE 24 (CIPK24).CBL1 requires dual lipid modification with N-myristoylation and S-acylation for localization to membrane microdomains (yellow section of plasma membrane) and subsequent activation of CIPK26, promoting reactive oxygen species accumulation in the cell wall via RESPIRATORY BURST OXIDASE HOMOLOG PROTEINS C and F (RBOH-C/F) contributing to cell wall loosening.

Figure 4 .
Figure 4. S-acylated proteins during CSC assembly and trafficking.CELLULOSE SYNTHASE A (CESA) proteins form the core of the CSC.A host of other proteins are known to either: colocalize with the CSC, associated with assembly and trafficking of the CSC to the plasma membrane (PM) or facilitate interactions between the cytoskeleton, the CSC and/or PM (reviewed in (Lampugnani et al. 2019; Polko and Kieber 2019; Gu and Rasmussen 2022).Many of these proteins are modified by S-acylation (indicated by red text).Proteins involved in cellulose synthesis illustrate the varied biological roles that S-acylation can play in protein function like membrane anchoring, subcellular trafficking and aiding the formation of protein complexes.S-acyl groups are shown for illustrative purposes and are indicated by a red zigzag line.The PM and cortical microtubule (CMT) are indicated together with proteins involved in cellulose synthesis of trafficking of the CSC.CMU, TPLATE Complex MUNISCIN-LIKE (TML), TRANSDUCIN/WD40 (TWD40), CSI, TARNVIA (TVA), MICROTUBULE-ASSOCIATED CELLULOSE SYNTHASE COMPARTMENT/Small CESA Compartments (MASC/SmaCC), KORRIGAN (KOR), TRANSMEMBRANE DOMAIN 5 (7TM5), ADAPTER PROTEIN COMPLEX 2 (AP2M), and COMPANION OF CELLULOSE SYNTHASE (CC) are indicated.